Transgenic plants are useful to study nuclear gene function and regulation and to improve agronomically important crop plants. Routine application of transgenic technology is made feasible by the alternative methods developed for transformation of nuclear genomes of higher plants. However, transgenic technology has not yet been applied to the genomes of the cytoplasmic organelles (i.e., plastids and mitochondria) of higher plants.
The size of plastid DNA (ptDNA) in higher plants is in the range of 120 kb to 160 kb (Palmer, Ann. Rev. Genet., 19: 325-54, 1985), and encodes the genes involved in plastid maintenance and photosynthesis. By now, three plastid genomes have been sequenced, including that of Nicotania tabacum (Shinozaki et al., EMBO J., 5: 2043-49, 1986). In addition to photosynthesis, plastids serve as a compartment for amino acid and lipid biosynthesis. Most if not all the genes involved in these functions are encoded by the nucleus. The enzymes encoded by nuclear genes are synthesized on cytoplasmic ribosomes, and are subsequently transported into the plastids. Expression and accumulation of nuclear gene products and of plastid gene products is coordinated (Gruissem, Cell, 56: 161-70, 1989).
Formation of stably transformed plastid genomes requires integration of the transforming DNA by recombination. That recombination is a mechanism contributing to the evolution of plastid genome is evident when comparing genomes of different plant species. Also, continued recombination through the plastid inverted repeat has been described (Palmer, 1985, supra). It has also been shown that intergenomic plastid recombination occurs in heteroplastidic cells obtained by protoplast fusion (Thanh & Medgyesy, Plant Mol. Biol. 12: 87-93, 1989), and is extensive (Fejes et al., Theor. App. Genet. 79: 28-32, 1990).
Introduction and stable integration of exogenous DNA has been reported recently in the plastid genome of a unicellular alga, Chlamydomonas reinhardtii (Boynton et al., Science 240: 1534-38, 1988; Blowers et al., Plant Cell 1: 123-32, 1989). Initial success in transforming the plastid genome of Chlamydomonas by Boynton et al. was made possible by the development of a microprojectile DNA delivery system based on a particle gun that delivers DNA-coated tungsten microprojectiles into the cell. In addition, a powerful selection scheme was applied that relied on complementing nonphotosynthetic deletion mutants. Subsequently, Blowers et al. showed that the Chlamydomonas plastid genome can be expanded by integrating the coding sequence of an E. coli enzyme, neomycin phosphotransferase. Transformation of the psbA gene encoding the D1 reaction center polypeptide of photosystem II, and of the 16s rRNA gene conferring resistance to streptomycin and spectinomycin was reported by Boynton et al., p.p. 509-16 in Current Research in Photosynthesis, M. Baltsheffsky, ed., 1990.
In higher plant chloroplasts, only transient expression of introduced DNA has been reported. DNA uptake and transient expression by isolated cucumber etioplasts of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans, or of the E. coli enzyme, chloramphenicol acetyltransferase (CAT), has been reported (Daniell and McFadden, Proc. Natl. Acad. Sci. USA 84: 6349-53, 1987). The 5' end of the psbA (pea), and rbcL (maize) plastid genes were fused with the CAT gene. Transient expression of the constructs in chloroplasts of cultured tobacco cells has been reported by Daniell et al., Proc. Natl. Acad. Sci. USA 87: 88-92 (1990) after biolistic delivery. Some of the vectors contained replication origins from ptDNA. CAT activity was sustained longer when the replicon origins were present. CAT activity, however, was not shown to be localized in chloroplasts. CAT activity, therefore, could have been the result of expression in the nucleus, since plastid gene promoters are known to support transcription initiation in the nucleus (Cornelissen and Vandewiele, Nucleic Acids Res. 17: 19-28, 1989).
European Application No. 87305573.5, filed Jun. 23, 1987, by M. C. Cannon and F. C. Cannon, describes a method for producing a plant whose cells express a desired gene by inserting the desired gene into the plastid genome of a plant cell. However, this application does not suggest a method for stably transforming plastids using nonlethal selection, nor does it provide any evidence suggesting that stable transformation was, or could be, achieved.
Transformation of plastids in higher plants was claimed after Agrobacterium-mediated transformation of N. tabacum (DeBlock et al., EMBO J. 4: 1367-72, 1985). A CAT gene was engineered for expression in the nucleus, and transgenic clones were selected for resistance to 10 .mu.g/ml chloramphenicol. The authors claim that there was fortuitous integration of the CAT gene into the ptDNA and expression from a plastid promoter. The line was reportedly unstable, and the authors' claims have not been confirmed.
There are several differences between the Chlamydomonas system and higher plants that may be relevant for successful transformation of plastids. Two of these are discussed below. First, the number of plastids, and the number of plastid genomes per cell is much lower in Chlamydomonas than in Nicotiana. Chlamydomonas contains a single plastid, which carries up to 80 identical plastid genomes (Harris, The Chlamydomonas Sourcebook, p. 354, Academic Press, San Diego, 1989). In contrast, Nicotiana tabacum cells contain a variable number of plastids, about 100 in leaf cells, and 12 to 14 in meristematic cells and dedifferentiated tissue culture cells (Thomas & Rose, Planta 158: 329-38, 1983). In a study with cultured cells the number of plastid genome copies was estimated to be 3,000 to 12,000 per cell (Cannon et al., Plant Cell Reports 4: 41-45, 1985; Yasuda et al., Planta 174: 235-41, 1988).
Another important difference is that Chlamydomonas cells are grown photoautotrophically which allows stringent selection for photosynthetic ability, that is, functional plastids. This facilitates transformation by all proven methods, including Agrobacterium-mediated transformation (Weising et al., Ann. Rev. Genet. 22: 421-77, 1988), electroporation (Fromm et al., Nature 319: 791-93, 1986), calcium phosphate coprecipitation (Krens et al., Nature 296: 72-74, 1982), transformation by high-velocity microprojectiles (Klein et al., Proc. Natl. Acad. Sci. USA 85: 8502-05, 1988), and polyethylene glycol treatment (Negrutiu et al., Plant Mol. Biol. 8: 363-73, 1987). In contrast, higher plants are cultured photoheterotrophically, which reduces the stringency of selection for functional plastids.
Given the large number of plastid genomes in plant cells, the ability to select for the transformed genome in culture is a key element in achieving plastid transformation. Selection markers have been identified by screening culture plant cells for mutants resistant to various substances, such as antibiotics and herbicides. Since most of the selectable plastid genome markers have been developed through cell culture, it is not surprising that most are derived from Nicotania tabacum and Nicotiana plumbaginifolia, two species that are easy to grow in cell culture and to subsequently regenerate into plants. Resistance to inhibitors of plastid protein synthesis, conferred by mutation in the plastid 16S rRNA and 23S rRNA genes, are the most readily available markers. Other markers include resistance to streptomycin (Maliga et al., Nature 255: 401-02, 1973; Etzold et al., FEBS Lett. 219: 343-46, 1987; Fromm et al., Plant Mol. Biol. 12: 499-505, 1989), spectinomycin (Fromm et al., EMBO J. 6: 3233-37, 1987) and lincomycin (Cseplo & Maliga, Mol. Gen. Genet. 196: 407-12, 1984; Cseplo et al., Mol. Gen. Genet. 214: 295-99, 1988) which are the equivalent rRNA gene mutation used for transformation in Chlamydomonas (Harris et al., 1989, supra).
Plastid genome mutants resistant to triazine herbicides, have also been obtained in cultured Nicotiana cells. Triazine herbicides inhibit photosynthesis by interruption of electron flow at the acceptor of photosystem II. Selection was made feasible in Nicotiana cultures by lowering the concentration of sucrose in the medium, thereby making cellular proliferation partially dependent on photosynthesis (photomyxotrophic cultures; Cseplo et al., Mol. Gen. Genet. 200: 508-10, 1985; Sato et al., Mol. Gen. Genet. 214: 358-60, 1988). Selection for resistance to this class of herbicides is also a nonlethal color selection, resistant mutants being identified by their green color (Cseplo et al., 1985, supra). A mutation in two of the lines was localized to the psbA gene (Pay et al., Nucleic Acids Res. 16: 8176, 1988; Sato et al., 1988, supra). Similar mutants have been found in higher plants under field conditions (Maliga et al., p.p. 133-143 in Perspectives in Genetic and Biochemical Regulation of Photosynthesis, I. Zeitlich, ed., Alan R. Liss, N.Y., 1990), and isolated in Chlamydomonas (Erickson et al., Science 228: 204-07, 1985).
Naturally occurring resistance to tentoxin is also encoded on the plastic genome (Durbin & Uchytil, Biochem. Genet. 15: 1143-45, 1977). Pigment deficiency caused by plastome mutation is frequent, but does not appear to be a useful marker in culture. Pigment mutation in combination with antibiotic resistance mutations, however, have proved important in recovering a recombinant plastid genome (Medgyesy et al., Proc. Natl. Acad. Sci. USA 82: 6960-64, 1985).
The present invention provides a method for stable transformation of the plastids of higher plants. Others have attempted to obtain stable plastid transformation in higher plants, but without success. The present invention also provides efficient and versatile DNA constructs to accomplish stable transformation of plastid genomes and expression of foreign proteins in transformed plastids. These methods and constructs, heretofore unavailable, will enable improvement of useful plant species by genetic engineering of the plastid genome.